Translational regulator

ABSTRACT

The invention provides a human translational regulator (TRANAC) and polynucleotides which identify and encode TRANAC. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating disorders associated with expression of TRANAC.

[0001] This application is a divisional application of pending U.S.application Ser. No. 09/215,063, filed on Dec. 17, 1998, which is adivisional application of U.S. application Ser. No. 08/869,733, filedJun. 5, 1997, now U.S. Pat. No. 5,955,278, issued Jun. 21, 1999, all ofwhich are entitled “New Translational Regulator,” the disclosure ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a new human translational regulator and to the use of these sequencesin the diagnosis, prevention, and treatment of inflammation anddisorders associated with cell proliferation and apoptosis.

BACKGROUND OF THE INVENTION

[0003] Protein synthesis is an indispensable process by which livingorganisms grow, differentiate, and propagate. The three stages ofprotein synthesis are initiation, elongation, and termination. Ineukaryotes, the first event in protein synthesis is the attachment of afree molecule of methionine (Met) to the end of a tRNA. Met-tRNA and asmall ribosomal RNA subunit 40S bind to the mRNA near the AUG initiationcodon to form the 40S complex. Addition of the 60S ribosomal RNA subunitto the 40S complex forms the peptidyl-tRNA transfer site. Once the mRNA,40S, and 60S complexes are in position, peptide synthesis may begin.

[0004] Translation initiation factors, eIF1a, eIF2, and eIF3, initiatethe formation of the 40S complex and are part of the complex as well.Binding between the 40S ribosomal RNA subunit and the mRNA is aided byeIF4a, eIF4b, and eIF4f using energy from the hydrolysis of GTP bound toeIF2. Translation initiation factor eIF5 promotes the hydrolysis ofribosome-bound GTP producing the energy necessary to bind the 40S and60S complexes.

[0005] eIF4f is a complex which recognizes 5′-mGpppN, the CAP structureof all eukaryotic mRNAs. The complex facilitates the association of the40S subunit to mRNA for translation initiation. Translation initiationis regulated by the phosphorylation of the CAP-binding protein, eIF4e, asubunit of the eIF4f complex. Addition of insulin to adipocytes ormuscle cells increases phosphorylation of eIF4e and stimulatesinitiation of protein synthesis (Morley, S. J. & Traugh, J. A. (1990) J.Biol. Chem. 265: 10611-10616). Overexpression of eIF4e has beenassociated with cell transformation (Lararis-Karatzas, A. et al. (1990)Nature 345: 544-547).

[0006] PHAS-I, PHAS-II and 4E-BP1 are three regulators of translationinitiation (Pause, A. et al. (1994) Nature 371: 762-767; Hu, C. et al.(1994) Proc. Natl. Acad. Sci. 91: 3730-3734; and Lin, T. -A. andLawrence, J. C. Jr. (1996) J. Biol. Chem 271: 30199-30204). Theassociation of PHAS-I or 4E-BP1 with eIF4e prevents the formation of theactive CAP-binding complex, eIF4f. Phosphorylation of PHAS-I or 4E-BP1by an insulin- or a growth factor-dependent kinase releases eIF4e from acomplex with PHAS-I or 4E-BP1 and prepares eIF4e to bind CAP fortranslation initiation. PHAS-I and PHAS-II are found to have overlappingbut different patterns of expression in tissues. Phosphorylation of bothPHAS proteins promotes dissociation of PHAS-eIF4e complexes andstimulates cell growth (Lin et al., supra).

[0007] The discovery of a new human translational regulator and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, prevention and treatmentof inflammation and disorders associated with cell proliferation andapoptosis.

SUMMARY OF THE INVENTION

[0008] The invention features a substantially purified polypeptide,human translational activator (TRANAC), having the amino acid sequenceshown in SEQ ID NO:1, or fragments thereof.

[0009] The invention further provides an isolated and substantiallypurified polynucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or fragments thereof. In a particularaspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:2 orvariants thereof.

[0010] In addition, the invention provides a polynucleotide sequencewhich hybridizes under stringent conditions to the polynucleotidesequence of SEQ ID NO:2. In another aspect the invention provides acomposition comprising an isolated and purified polynucleotide sequenceencoding TRANAC.

[0011] The invention further provides a polynucleotide sequencecomprising the complement of the polynucleotide sequence encoding theamino acid sequence of SEQ ID NO:1, or fragments or variants thereof. Ina particular aspect, the polynucleotide sequence is the complement ofSEQ ID NO:2. In another aspect the invention provides a compositioncomprising an isolated and purified polynucleotide sequence comprisingthe complement of SEQ ID NO:2, or fragments or variants thereof.

[0012] The present invention further provides an expression vectorcontaining at least a fragment of any of the claimed polynucleotidesequences. In yet another aspect, the expression vector containing thepolynucleotide sequence is contained within a host cell.

[0013] The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding TRANAC under conditions suitable forthe expression of the polypeptide; and b) recovering the polypeptidefrom the host cell culture.

[0014] The invention also provides a pharmaceutical compositioncomprising a substantially purified TRANAC having the amino acidsequence of SEQ ID NO:1 in conjunction with a suitable pharmaceuticalcarrier.

[0015] The invention also provides a purified antagonist which decreasesthe activity of a polypeptide of SEQ ID NO:1. In one aspect, theinvention provides a purified antibody which binds to a polypeptidecomprising at least a fragment of the amino acid sequence of SEQ IDNO:1.

[0016] Still further, the invention provides a purified agonist whichmodulates the activity of the polypeptide of SEQ ID NO:1.

[0017] The invention also features a method for treating or preventinginflammation by administering TRANAC, a method for treating orpreventing cancer by administering TRANAC, a method for treating orpreventing an disorder associated with apoptosis by administering anantagonist of TRANAC, and a method for stimulating cell proliferation byadministering an antagonist of TRANAC.

[0018] The invention also provides a method for detecting apolynucleotide which encodes TRANAC in a biological sample comprisingthe steps of: a) hybridizing a polynucleotide sequence complementary toTRANAC (SEQ ID NO:1) to nucleic acid material of a biological sample,thereby forming a hybridization complex; and b) detecting thehybridization complex, wherein the presence of the complex correlateswith the presence of a polynucleotide encoding TRANAC in the biologicalsample. In a preferred embodiment, prior to hybridization, the nucleicacid material of the biological sample is amplified by the polymerasechain reaction.

BRIEF DESCRIPTION OF THE FIGURES

[0019]FIG. 1A and 1B show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of TRANAC. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.Ltd. San Bruno, Calif.).

[0020]FIG. 2 shows the amino acid sequence alignments among TRANAC (SEQID NO:1) and a human translational regulator, 4E-BP1 (GI 561632; SEQ IDNO:3), and a mouse translational regulator, PHAS-II (GI 1658516; SEQ IDNO:4), produced using the multisequence alignment program of DNASTAR™software (DNASTAR Inc, Madison Wis.).

[0021]FIGS. 3A and 3B show the hydrophobicity plots for TRANAC, SEQ IDNO:1 and 4E-BP1 (SEQ ID NO:3), respectively. The positive X axisreflects amino acid position, and the negative Y axis, hydrophobicity(MACDNASIS PRO software).

DESCRIPTION OF THE INVENTION

[0022] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0023] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0024] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0025] Definitions

[0026] TRANAC, as used herein, refers to the amino acid sequences ofsubstantially purified TRANAC obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0027] The term “agonist”, as used herein, refers to a molecule which,when bound to TRANAC, increases or prolongs the duration of the effectof TRANAC. Agonists may include proteins, nucleic acids, carbohydrates,or any other molecules which bind to and modulate the effect of TRANAC.

[0028] An “allele” or “allelic sequence”, as used herein, is analternative form of the gene encoding TRANAC. Alleles may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given natural or recombinant gene may have none, one, ormany allelic forms. Common mutational changes which give rise to allelesare generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0029] “Altered” nucleic acid sequences encoding TRANAC as used hereininclude those with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent TRANAC. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding TRANAC,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding TRANAC. The encoded protein may also be “altered” and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent TRANAC.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of TRANAC is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

[0030] “Amino acid sequence” as used herein refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules. Fragments of TRANAC arepreferably about 5 to about 15 amino acids in length and retain thebiological activity or the immunological activity of TRANAC. Where“amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, amino acid sequence,and like terms, are not meant to limit the amino acid sequence to thecomplete, native amino acid sequence associated with the recited proteinmolecule.

[0031] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0032] The term “antagonist” as used herein, refers to a molecule which,when bound to TRANAC, decreases the amount or the duration of the effectof the biological or immunological activity of TRANAC. Antagonists mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich and decrease the effect of TRANAC.

[0033] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind TRANACpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or oligopeptide used to immunize an animal can be derivedfrom the translation of RNA or synthesized chemically and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin, keyhole limpet hemocyanin. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

[0034] The term “antigenic determinant”, as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0035] The term “antisense”, as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

[0036] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic TRANAC, orany oligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0037] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

[0038] A “composition comprising a given polynucleotide sequence” asused herein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding TRANAC (SEQ ID NO:1) or fragments thereof (e.g., SEQ ID NO:2and fragments thereof) may be employed as hybridization probes. Theprobes may be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

[0039] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, has been extendedusing XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5′ and/or the 3′direction and resequenced, or has been assembled from the overlappingsequences of more than one Incyte Clone using a computer program forfragment assembly (e.g., GELVIEW™ Fragment Assembly system, GCG,Madison, Wis.). Some sequences have been both extended and assembled toproduce the consensus sequence.

[0040] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 by northern analysis is indicativeof the presence of mRNA encoding TRANAC in a sample and therebycorrelates with expression of the transcript from the polynucleotideencoding the protein.

[0041] A “deletion”, as used herein, refers to a change in the aminoacid or nucleotide sequence and results in the absence of one or moreamino acid residues or nucleotides.

[0042] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to TRANAC orthe encoded TRANAC. Such modifications include, for example, replacementof hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

[0043] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

[0044] Human artificial chromosomes (HACs) are linear microchromosomeswhich may contain DNA sequences of 10K to 10M in size and contain all ofthe elements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

[0045] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0046] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0047] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

[0048] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

[0049] “Microarray” refers to a high-density array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support.

[0050] The term “modulate”, as used herein, refers to a change in theactivity of TRANAC. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional or immunological properties of TRANAC.

[0051] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments thereof,and to DNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand.“Fragments” are those nucleic acid sequences which are greater than 60nucleotides than in length, and most preferably includes fragments thatare at least 100 nucleotides or at least 1000 nucleotides, and at least10,000 nucleotides in length.

[0052] The term “oligonucleotide” refers to a nucleic acid sequence ofat least about 6 nucleotides to about 60 nucleotides, preferably about15 to 30 nucleotides, and more preferably about 20 to 25 nucleotides,which can be used in PCR amplification or hybridization assays. As usedherein, oligonucleotide is substantially equivalent to the terms“amplimers”, “primers”, “oligomers”, and “probes”, as commonly definedin the art.

[0053] “Peptide nucleic acid”, PNA as used herein, refers to anantisense molecule or anti-gene agent which comprises an oligonucleotideof at least five nucleotides in length linked to a peptide backbone ofamino acid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

[0054] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from five amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length TRANAC and fragments thereof.

[0055] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encodingTRANAC, or fragments thereof, or TRANAC itself may comprise a bodilyfluid, extract from a cell, chromosome, organelle, or membrane isolatedfrom a cell, a cell, genomic DNA, RNA, or cDNA(in solution or bound to asolid support, a tissue, a tissue print, and the like.

[0056] The terms “specific binding” or “specifically binding”, as usedherein, refers to that interaction between a protein or peptide and anagonist, an antibody and an antagonist. The interaction is dependentupon the presence of a particular structure (i.e., the antigenicdeterminant or is epitope) of the protein recognized by the bindingmolecule. For example, if an antibody is specific for epitope “A”, thepresence of a protein containing epitope A (or free, unlabeled A) in areaction containing labeled “A” and the antibody will reduce the amountof labeled A bound to the antibody.

[0057] The terms “stringent conditions” or “stringency”, as used herein,refer to the conditions for hybridization as defined by the nucleicacid, salt, and temperature. These conditions are well known in the artand may be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

[0058] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0059] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0060] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0061] A “variant” of TRANAC, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Analogousminor variations may also include amino acid deletions or insertions, orboth. Guidance in determining which amino acid residues may besubstituted, inserted, or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNASTAR software.

[0062] The Invention

[0063] The invention is based on the discovery of a new humantranslational regulator (hereinafter referred to as “TRANAC”), thepolynucleotides encoding TRANAC, and the use of these compositions forthe diagnosis, prevention, or treatment of inflammation and disordersassociated with cell proliferation and apoptosis.

[0064] Nucleic acids encoding the TRANAC of the present invention werefirst identified in Incyte Clone 805296 from brain stem tissue cDNAlibrary (BSTMNOT01) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:2, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 882099 (THYRNOT02), 805296 (BSTMNOT01), 636579 (NEUTGMT01), and1254309 (LUNGFET03).

[0065] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1Aand 1B. TRANAC is 100 amino acids in length and has high abundance ofproline, serine, threonine, and glycine. TRANAC also has two potentialcasein kinase II phosphorylation sites encompassing residues S76-E79 andT86-E89. As shown in FIG. 2, TRANAC has chemical and structural homologywith 4E-BP1 (GI 561632; SEQ ID NO:3) and PHAS-II (GI 1658516; SEQ IDNO:4). In particular, TRANAC shares 60% and 57% identity with 4E-BP1 andPHAS-II, respectively. As illustrated by FIGS. 3A and 3B, TRANAC and4E-BP1 have rather similar hydrophobicity plots. Northern analysis showsthe expression of this sequence in various libraries, at least 42% ofwhich are immortalized or cancerous, at least 12% of which involveimmune response, and at least 15% of which involve infant/fetal tissuesor organs.

[0066] The invention also encompasses TRANAC variants. A preferredTRANAC variant is one having at least 80%, and more preferably 90%,amino acid sequence identity to the TRANAC amino acid sequence (SEQ IDNO:1). A most preferred TRANAC variant is one having at least 95% aminoacid sequence identity to SEQ ID NO:1.

[0067] The invention also encompasses polynucleotides which encodeTRANAC. Accordingly, any nucleic acid sequence which encodes the aminoacid sequence of TRANAC can be used to produce recombinant moleculeswhich express TRANAC. In a particular embodiment, the inventionencompasses the polynucleotide comprising the nucleic acid sequence ofSEQ ID NO:2 as shown in FIGS. 1A-C.

[0068] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding TRANAC, some bearing minimal homology to thenucleotide sequences of any known and naturally occurring gene, may beproduced. Thus, the invention contemplates each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring TRANAC, and all suchvariations are to be considered as being specifically disclosed.

[0069] Although nucleotide sequences which encode TRANAC and itsvariants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring TRANAC under appropriately selectedconditions of stringency, it may be advantageous to produce nucleotidesequences encoding TRANAC or its derivatives possessing a substantiallydifferent codon usage. Codons may be selected to increase the rate atwhich expression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding TRANAC and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0070] The invention also encompasses production of DNA sequences, orfragments thereof, which encode TRANAC and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding TRANAC or any fragment thereof.

[0071] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2, under various conditions ofstringency as taught in Wahi, G. M. and S. L. Berger (1987; MethodsEnzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-511).

[0072] Methods for DNA sequencing which are well known and generallyavailable in the art and may be used to practice any of the embodimentsof the invention. The methods may employ such enzymes as the Klenowfragment of DNA polymerase I, Sequenase® (US Biochemical Corp,Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7polymerase (Amersham, Chicago, Ill.), or combinations of polymerases andproofreading exonucleases such as those found in the ELONGASEAmplification System marketed by Gibco/BRL (Gaithersburg, Md.).Preferably, the process is automated with machines such as the HamiltonMicro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI Catalyst and 373 and 377 DNASequencers (Perkin Elmer).

[0073] The nucleic acid sequences encoding TRANAC may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

[0074] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

[0075] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

[0076] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions. When screening for full-length cDNAs, it ispreferable to use libraries that have been size-selected to includelarger cDNAs. Also, random-primed libraries are preferable, in that theywill contain more sequences which contain the 5′ regions of genes. Useof a randomly primed library may be especially preferable for situationsin which an oligo d(T) library does not yield a full-length cDNA.Genomic libraries may be useful for extension of sequence into 5′non-transcribed regulatory regions.

[0077] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled devise camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. Genotyper™ andSequence Navigator™, Perkin Elmer) and the entire process from loadingof samples to computer analysis and electronic data display may becomputer controlled. Capillary electrophoresis is especially preferablefor the sequencing of small pieces of DNA which might be present inlimited amounts in a particular sample.

[0078] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode TRANAC may be used in recombinant DNAmolecules to direct expression of TRANAC, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressTRANAC.

[0079] As will be understood by those of skill in the art, it may beadvantageous to produce TRANAC-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

[0080] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterTRANAC encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0081] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding TRANAC may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of TRANAC activity, it may be useful toencode a chimeric TRANAC protein that can be recognized by acommercially available antibody. A fusion protein may also be engineeredto contain a cleavage site located between the TRANAC encoding sequenceand the heterologous protein sequence, so that TRANAC may be cleaved andpurified away from the heterologous moiety.

[0082] In another embodiment, sequences encoding TRANAC may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of TRANAC, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

[0083] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of TRANAC, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0084] In order to express a biologically active TRANAC, the nucleotidesequences encoding TRANAC or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0085] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encodingTRANAC and appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described in Sambrook, J. et al. (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., andAusubel, F. M. et al. (1989) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y.

[0086] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding TRANAC. These include, but arenot limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems. The invention is not limited by the host cell employed.

[0087] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding TRANAC,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0088] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for TRANAC. For example, whenlarge quantities of TRANAC are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding TRANAC may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0089] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0090] In cases where plant expression vectors are used, the expressionof sequences encoding TRANAC may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

[0091] An insect system may also be used to express TRANAC. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encodingTRANAC may be cloned into a non-essential region of the virus, such asthe polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of TRANAC will render the polyhedrin geneinactive and produce recombinant virus lacking coat protein. Therecombinant viruses may then be used to infect, for example, S.frugiperda cells or Trichoplusia larvae in which TRANAC may be expressed(Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0092] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding TRANAC may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing TRANAC in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0093] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

[0094] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding TRANAC. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding TRANAC, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0095] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0096] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress TRANAC may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0097] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0098] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding TRANACis inserted within a marker gene sequence, transformed cells containingsequences encoding TRANAC can be identified by the absence of markergene function. Alternatively, a marker gene can be placed in tandem witha sequence encoding TRANAC under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0099] Alternatively, host cells which contain the nucleic acid sequenceencoding TRANAC and express TRANAC may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0100] The presence of polynucleotide sequences encoding TRANAC can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding TRANAC.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding TRANAC todetect transformants containing DNA or RNA encoding TRANAC.

[0101] A variety of protocols for detecting and measuring the expressionof TRANAC, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson TRANAC is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0102] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encodingTRANAC include oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding TRANAC, or any fragments thereof may be cloned into a vectorfor the production of an mRNA probe. Such vectors are known in the art,are commercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Minn.); Promega (Madison, Wis.); and U.S. Biochemical Corp., Cleveland,Ohio). Suitable reporter molecules or labels, which may be used for easeof detection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

[0103] Host cells transformed with nucleotide sequences encoding TRANACmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode TRANAC may be designed to contain signal sequences which directsecretion of TRANAC through a prokaryotic or eukaryotic cell membrane.Other constructions may be used to join sequences encoding TRANAC tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and TRANAC may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingTRANAC and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying TRANAC from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

[0104] In addition to recombinant production, fragments of TRANAC may beproduced by direct peptide synthesis using solid-phase techniquesMerrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using an Applied Biosystems 431Apeptide synthesizer (Perkin Elmer). Various fragments of TRANAC may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

[0105] Therapeutics

[0106] Chemical and structural homology exists between TRANAC and twotranslational regulators, 4E-BP1 (GI 561632) and PHAS-II (GI 1658516).Northern analysis shows that the expression of TRANAC is associated withcell proliferation, fetal and infant development, inflammation, andimmune response.

[0107] Decreased expression of TRANAC appears to be associated withincreased cell proliferation. Therefore, in one embodiment, TRANAC or afragment or derivative thereof may be administered to a subject toprevent or treat a disorder associated with excessive cellproliferation. Such disorders include various types of cancer including,but not limited to, adenocarcinoma, leukemia, lymphoma, melanoma,myeloma, sarcoma, and teratocarcinoma, and particularly, cancers of theadrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,skin, spleen, testis, thymus, thyroid, and uterus.

[0108] In another embodiment, an agonist of TRANAC or a derivative orfragment thereof may be used to modulate the activity of TRANAC and toprevent or treat a disorder associated with excessive cell proliferationincluding, but not linited to, those listed above.

[0109] In still another embodiment, a vector capable of expressingTRANAC, or a fragment or a derivative thereof, may be used to prevent ortreat a disorder associated with excessive cell proliferation including,but not limited to, those listed above.

[0110] In another embodiment, TRANAC or a fragment or derivative thereofmay be administered to a subject to prevent or treat inflammationassociated with any disorder of immune response including, but are notlimited to, Addison's disease, adult respiratory distress syndrome,allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitus,Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma.

[0111] In another embodiment, an agonist of TRANAC or a fragment orderivative thereof may be used to modulate the activity of TRANAC and toprevent or treat inflammation associated with any disorder including,but not limited to, those listed above.

[0112] In still another embodiment, a vector capable of expressingTRANAC, or a fragment or a derivative thereof, may be used to prevent ortreat inflammation associated with any disorder including, but notlimited to, those listed above.

[0113] Increased expression of TRANAC results in apoptosis in normalfetal development. However, increased expression of TRANAC in othersubjects may result in apoptosis which may have detrimental effects.Therefore, in one embodiment, an antagonist of TRANAC or a fragment orderivative thereof may be administered to a subject to prevent or treata disorder with associated apoptosis. Such disorders include, but arenot limited to, AIDS and other infectious or genetic immunodeficiencies,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, retinitis pigmentosa, andcerebellar degeneration, myelodysplastic syndromes such as aplasticanemia, ischemic injuries such as myocardial infarction, stroke, andreperfusion injury, toxin-induced diseases such as alcohol-induced liverdamage, cirrhosis, and lathyrism, wasting diseases such as cachexia,viral infections such as by hepatitis B and C, and osteoporosis. In oneaspect, an antibody specific for TRANAC may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express TRANAC.

[0114] In another embodiment, a vector expressing the antisense orcomplementary sequence of the polynucleotide encoding TRANAC, or afragment or a derivative thereof, may be used to prevent or treat adisorder associated with increased apoptosis including, but not limitedto, those listed above.

[0115] In a further embodiment, an antagonist or an inhibitor of TRANAC,or a fragment or a derivative thereof, may be added to cells tostimulate cell proliferation. In particular, an antagonist of TRANAC maybe added to a cell or cells in vivo using delivery mechanisms such asliposomes, viral based vectors, or electroinjection for the purpose ofpromoting regeneration or differentiation of the cell or cells. Inaddition, an antagonist of TRANAC may be added to a cell, cell line,tissue or organ culture in vitro or ex vivo to stimulate cellproliferation for use in heterologous or autologous transplantation. Insome cases, the cell will have been selected for its ability to fight aninfection or a cancer or to correct a genetic defect such as sickle cellanemia, β thalassemia, cystic fibrosis, or Huntington's chorea. In oneaspect, an antibody specific for TRANAC may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express TRANAC.

[0116] In a still further embodiment, a vector expressing the antisenseor complementary sequence of the polynucleotide encoding TRANAC, or afragment or a derivative thereof, may be used to stimulate cellproliferation, as detailed above.

[0117] In other embodiments, any of the therapeutic proteins,antagonists, antibodies, agonists, complementary sequences or vectors ofthe invention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0118] Antagonists or inhibitors of TRANAC may be produced using methodswhich are generally known in the art. In particular, purified TRANAC maybe used to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind TRANAC.

[0119] Antibodies to TRANAC may be generated using methods that are wellknown in the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0120] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith TRANAC or any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0121] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to TRANAC have an amino acid sequenceconsisting of at least five amino acids and more preferably at least 10amino acids. It is also preferable that they are identical to a portionof the amino acid sequence of the natural protein, and they may containthe entire amino acid sequence of a small, naturally occurring molecule.Short stretches of TRANAC amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0122] Monoclonal antibodies to TRANAC may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0123] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S.L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceTRANAC-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0124] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0125] Antibody fragments which contain specific binding sites forTRANAC may also be generated. For example, such fragments include, butare not limited to, the F(ab′)2 fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)2fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0126] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between TRANAC and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering TRANAC epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0127] In another embodiment of the invention, the polynucleotidesencoding TRANAC, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding TRANAC may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding TRANAC. Thus, complementary molecules orfragments may be used to modulate TRANAC activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments, can bedesigned from various locations along the coding or control regions ofsequences encoding TRANAC.

[0128] Expression vectors derived from retro viruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encodingTRANAC. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

[0129] Genes encoding TRANAC can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes TRANAC. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0130] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′ or regulatory regions of the geneencoding TRANAC (signal sequence, promoters, enhancers, and introns).Oligonucleotides derived from the transcription initiation site, e.g.,between positions −10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0131] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding TRANAC.

[0132] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0133] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding TRANAC. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA constitutively or inducibly can be introduced into cell lines,cells, or tissues.

[0134] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0135] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposome injectionsor polycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

[0136] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0137] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of TRANAC,antibodies to TRANAC, mimetics, agonists, antagonists, or inhibitors ofTRANAC. The compositions may be administered alone or in combinationwith at least one other agent, such as stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0138] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0139] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0140] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0141] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0142] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0143] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0144] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Non-lipid polycationic amino polymers may also be used for delivery.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0145] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0146] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0147] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0148] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of TRANAC, such labeling wouldinclude amount, frequency, and method of administration.

[0149] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0150] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0151] A therapeutically effective dose refers to that amount of activeingredient, for example TRANAC or fragments thereof, antibodies ofTRANAC, agonists, antagonists or inhibitors of TRANAC, which amelioratesthe symptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0152] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0153] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0154] Diagnostics

[0155] In another embodiment, antibodies which specifically bind TRANACmay be used for the diagnosis of conditions or diseases characterized byexpression of TRANAC, or in assays to monitor patients being treatedwith TRANAC, agonists, antagonists or inhibitors. The antibodies usefulfor diagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for TRANAC includemethods which utilize the antibody and a label to detect TRANAC in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0156] A variety of protocols including ELISA, RIA, and FACS formeasuring TRANAC are known in the art and provide a basis for diagnosingaltered or abnormal levels of TRANAC expression. Normal or standardvalues for TRANAC expression are established by combining body fluids orcell extracts taken from normal mammalian subjects, preferably human,with antibody to TRANAC under conditions suitable for complex formation.The amount of standard complex formation may be quantified by variousmethods, but preferably by photometric, means. Quantities of TRANACexpressed in subject, control and disease, samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0157] In another embodiment of the invention, the polynucleotidesencoding TRANAC may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof TRANAC may be correlated with disease. The diagnostic assay may beused to distinguish between absence, presence, and excess expression ofTRANAC, and to monitor regulation of TRANAC levels during therapeuticintervention.

[0158] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding TRANAC or closely related molecules, may be used to identifynucleic acid sequences which encode TRANAC. The specificity of theprobe, whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding TRANAC, alleles, or related sequences.

[0159] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the TRANAC encoding sequences. The hybridization probes of thesubject invention may be DNA or RNA and derived from the nucleotidesequence of SEQ ID NO:2 or from genomic sequence including promoter,enhancer elements, and introns of the naturally occurring TRANAC.

[0160] Means for producing specific hybridization probes for DNAsencoding TRANAC include the cloning of nucleic acid sequences encodingTRANAC or TRANAC derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, commercially available, andmay be used to synthesize RNA probes in vitro by means of the additionof the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, radionuclides such as 32P or 35S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0161] Polynucleotide sequences encoding TRANAC may be used for thediagnosis of conditions, disorders, or diseases which are associatedwith expression of TRANAC. Examples of such disorders include: varioustypes of cancer such as adenocarcinoma, leukemia, lymphoma, melanoma,myeloma, sarcoma, and teratocarcinoma, and particularly, cancers of theadrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,skin, spleen, testis, thymus, thyroid, and uterus; disorders associatedwith inflammation such as Addison's disease, adult respiratory distresssyndrome, allergies, anemia, asthma, atherosclerosis, bronchitis,cholecystitus, Crohn's disease, ulcerative colitis, atopic dermatitis,dermatomyositis, diabetes mellitus, emphysema, atrophic gastritis,glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjögren's syndrome, and autoimmune thyroiditis;complications of cancer, hemodialysis, extracorporeal circulation;viral, bacterial, fungal, parasitic, protozoal, and helminthicinfections and trauma; and disorders associated apoptosis such as AIDSand other infectious or genetic immunodeficiencies, neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, amyotrophiclateral sclerosis, retinitis pigmentosa, and cerebellar degeneration,myelodysplastic syndromes such as aplastic anemia, ischemic injuriessuch as myocardial infarction, stroke, and reperfusion injury,toxin-induced diseases such as alcohol-induced liver damage, cirrhosis,and lathyrism, wasting diseases such as cachexia, viral infections suchas by hepatitis B and C, and osteoporosis. The polynucleotide sequencesencoding TRANAC may be used in Southern or northern analysis, dot blot,or other membrane-based technologies; in PCR technologies; or indipstick, pin, ELISA assays or microarrays utilizing fluids or tissuesfrom patient biopsies to detect altered TRANAC expression. Suchqualitative or quantitative methods are well known in the art.

[0162] In a particular aspect, the nucleotide sequences encoding TRANACmay be useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding TRANAC may be labeled by standard methods, and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding TRANAC in the sample indicates thepresence of the associated disease. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0163] In order to provide a basis for the diagnosis of diseaseassociated with expression of TRANAC, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes TRANAC,under conditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0164] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0165] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0166] Additional diagnostic uses for oligonucleotides designed from thesequences encoding TRANAC may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5′->3′) and another with antisense (3′<-5′),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

[0167] Methods which may also be used to quantitate the expression ofTRANAC include radiolabeling or biotinylating nucleotides,coamplification of a control nucleic acid, and standard curves ontowhich the experimental results are interpolated (Melby, P. C. et al.(1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal.Biochem. 229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0168] In further embodiments, oligonucleotides derived from any of thepolynucleotide sequences described herein may be used as probes inmicroarrays. The microarrays can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents.

[0169] In one embodiment, the microarray is prepared and used accordingto the methods described in PCT application WO95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference.

[0170] The microarray is preferably composed of a large number ofunique, single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs fixed to a solidsupport. Microarrays may contain oligonucleotides which cover the known5′, or 3′, sequence, or contain sequential oligonucleotides which coverthe full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state.

[0171] In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5′ or more preferably at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. The oligomers are synthesized atdesignated areas on a substrate using a light-directed chemical process.The substrate may be paper, nylon or other type of membrane, filter,chip, glass slide or any other suitable solid support.

[0172] In another aspect, the oligomers may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available devises (slot blot or dot blotapparatus) materials and machines (including robotic instruments) andcontain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144dots, or any other multiple which lends itself to the efficient use ofcommercially available instrumentation.

[0173] In order to conduct sample analysis using the microarrays, theRNA or DNA from a biological sample is made into hybridization probes.The mRNA is isolated, and cDNA is produced and used as a template tomake antisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies on the sequences, mutations, variants, or polymorphisms amongsamples.

[0174] In another embodiment of the invention, the nucleic acidsequences which encode TRANAC may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome, to aspecific region of a chromosome or to artificial chromosomeconstructions, such as human artificial chromosomes (HACs), yeastartificial chromosomes (YACs), bacterial artificial chromosomes (BACs),bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0175] Fluorescent in situ hybridization (FISH as described in Verma etal. (1988) Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York, N.Y.) may be correlated with other physical chromosomemapping techniques and genetic map data. Examples of genetic map datacan be found in various scientific journals or at Online MendelianInheritance in Man (OMIM). Correlation between the location of the geneencoding TRANAC on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

[0176] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0177] In another embodiment of the invention, TRANAC, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenTRANAC and the agent being tested, may be measured.

[0178] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to TRANAC largenumbers of different small test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with TRANAC, or fragments thereof, and washed.Bound TRANAC is then detected by methods well known in the art. PurifiedTRANAC can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

[0179] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding TRANACspecifically compete with a test compound for binding TRANAC. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with TRANAC.

[0180] In additional embodiments, the nucleotide sequences which encodeTRANAC may be used in any molecular biology techniques that have yet tobe developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including, but notlimited to, such properties as the triplet genetic code and specificbase pair interactions.

[0181] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0182] I BSTMNOT01 cDNA Library Construction

[0183] The BSTMNOT01 cDNA library was constructed from normal brain stemtissue. The donor was a 72 year old male who had died of a myocardialinfaction (specimen #RA95-05-0323; International Institute for AdvancedMedicine, Exton, Pa.).

[0184] Cryopreserved tissue was homogenized and lysed using a BrinkmannPOLYTRON PT-3000 Homogenizer (Brinkmann Instruments, Westbury, N.J.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with phenol chloroform pH8.0, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water and DNase treated at 37° C. Extractionand precipitation were repeated as before. The mRNA was then isolatedusing the QIAGEN OLIGOTEX mRNA purification kit (QIAGEN, Chatsworth,Calif.) and used to construct the cDNA library.

[0185] The mRNA was handled according to the recommended protocols inthe SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning(Cat. #18248-013; Gibco/BRL). cDNAs were fractionated on a SepharoseCL4B column (Cat. #275105-01; Pharmacia), and those cDNAs exceeding 400bp were ligated into pSport I. The plasmid pSport I was subsequentlytransformed into DH5α competent cells (Cat. #18258-012; Gibco/BRL).

[0186] II Isolation and Sequencing of cDNA Clones

[0187] Plasmid DNA was released from the cells and purified using theR.E.A.L. PREP 96 plasmid purification kit (Catalog #26173; QIAGEN). Thiskit enables the simultaneous purification of 96 samples in a 96-wellblock using multi-channel reagent dispensers. The recommended protocolwas employed except for the following changes: 1) the bacteria werecultured in 1 ml of sterile Terrific Broth (Catalog #22711, Gibco/BRL)with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) afterinoculation, the cultures were incubated for 19 hours after the wellsand at the end of the incubation, the cells were lysed with 0.3 ml oflysis buffer; and 3) following isopropanol precipitation, the plasmidDNA pellet was resuspended in 0.1 ml of distilled water. After the laststep in the protocol, samples were transferred to a 96-well block forstorage at 4° C.

[0188] The cDNAs were sequenced by the method of Sanger F and A RCoulson (1975; J Mol Biol 94:441f), using a Hamilton MICROLAB 2200liquid transfer system (Hamilton, Reno, Nev.) in combination withPeltier Thermal Cyclers (PTC200 from MJ Research, Watertown, Mass.) andApplied Biosystems' 377 DNA sequencing systems (Applied Biosystems,Foster City, Calif.); and the reading frame was determined.

[0189] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0190] The nucleotide sequences of the Sequence Listing as well as theamino acid sequences deduced from them were used as query sequencesagainst databases such as GenBank, SwissProt, BLOCKS, and Pima II. Thesedatabases, which contain previously identified and annotated sequences,were searched for regions of homology (similarity) using BLAST, whichstands for Basic Local Alignment Search Tool (Altschul, S. F (1993) J.Mol. Evol. 36: 290-300; Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410).

[0191] BLAST produced alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST was especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal, or plant) origin. Otheralgorithms such as the one described in Smith et al. (1992, ProteinEngineering 5:35-51), incorporated herein by reference, could have beenused when dealing with primary sequence patterns and secondary structuregap penalties. The sequences disclosed in this application have lengthsof at least 49 nucleotides, and no more than 12% uncalled bases (where Nis recorded rather than A, C, G, or T).

[0192] The BLAST approach, as detailed in Karlin, S. and Altschul, S. F.(1993) Proc. Natl. Acad. Sci. 90: 5873-5877 and incorporated herein byreference, searched for matches between a query sequence and a databasesequence. BLAST evaluated the statistical significance of any matchesfound, and reported only those matches that satisfy the user-selectedthreshold of significance. In this application, threshold was set at10⁻²⁵ for nucleotides and 10⁻¹⁴ for peptides.

[0193] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and other mammalian sequences(mam); and deduced amino acid sequences from the same clones were thensearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp) for homology. The relevantdatabase for a particular match were reported as Glxxx±p (where xxx ispri, rod, etc., and if present, p=peptide).

[0194] IV Northern Analysis

[0195] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0196] Analogous computer techniques using BLAST (Altschul, S. F. 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or the LIFESEQ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0197] The basis of the search is the product score which is defined as:

% sequence identity x % maximum BLAST score/100

[0198] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0199] The results of northern analysis are reported as a list oflibraries in which the transcript encoding TRANAC occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0200] V Extension of TRANAC Encoding Polynucleotides

[0201] The nucleic acid sequence of the Incyte Clone 805296 was used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length. One primer was synthesized to initiateextension in the antisense direction, and the other was synthesized toextend sequence in the sense direction. Primers were used to facilitatethe extension of the known sequence “outward” generating ampliconscontaining new, unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO 4.06 primeranalysis software (National Biosciences), or another appropriateprogram, to be about 22 to about 30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures of about 68° to about 72° C. Any stretch of nucleotideswhich would result in hairpin structures and primer-primer dimerizationswas avoided.

[0202] Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0203] High fidelity amplification was obtained by following theinstructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing theenzyme and reaction mix. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR wasperformed using the PTC-200 thermal cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (and holding)

[0204] A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQUICK PCR purification kit (QIAGEN Inc., Chatsworth,Calif.), and trimmed of overhangs using Klenow enzyme to facilitatereligation and cloning.

[0205] After ethanol precipitation, the products were redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2×Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2×Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

[0206] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

[0207] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid, and sequenced.

[0208] In like manner, the nucleotide sequence of SEQ ID NO:2 is used toobtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for 5′ extension, and an appropriate genoliclibrary.

[0209] VI Labeling and Use of Individual Hybridization Probes

[0210] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 primer analysis software (National Biosciences),labeled by combining 50 pmol of each oligomer and 250 μCi of [γ-³²P]adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPontNEN®, Boston, Mass.). The labeled oligonucleotides are substantiallypurified with Sephadex G-25 superfine resin column (Pharmacia & Upjohn).A aliquot containing 10⁷ counts per minute of the labeled probe is usedin a typical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases (Ase I, Bgl II, Eco RI,Pst I, Xba 1, or Pvu II; DuPont NEN®).

[0211] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

[0212] VII Microarrays

[0213] To produce oligonucleotides for a micro array, the nucleotidesequence described herein is examined using a computer algorithm whichstarts at the 3′ end of the nucleotide sequence. The algorithmidentifies oligomers of defined length that are unique to the gene, havea GC content within a range suitable for hybridization, and lackpredicted secondary structure that would interfere with hybridization.The algorithm identifies 20 sequence-specific oligonucleotides of 20nucleotides in length (20-mers). A matched set of oligonucleotides iscreated in which one nucleotide in the center of each sequence isaltered. This process is repeated for each gene in the microarray, anddouble sets of twenty 20 mers are synthesized and arranged on thesurface of the silicon chip using a light-directed chemical process(Chee, M. et al., PCT/WO95/11995, incorporated herein by reference).

[0214] In the alternative, a chemical coupling procedure and an ink jetdevice are used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCT/WO95/25116, incorporated herein byreference). In another alternative, a “gridded” array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

[0215] VIII Complementary Polynucleotides

[0216] Sequence complementary to the TRANAC-encoding sequence, or anypart thereof, is used to decrease or inhibit expression of naturallyoccurring TRANAC. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 primer analysis softwareand the coding sequence of TRANAC, SEQ ID NO:1. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the TRANAC-encoding transcript.

[0217] IX Expression of TRANAC

[0218] Expression of TRANAC is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express TRANAC in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0219] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of TRANAC into the bacterial growth media which can be useddirectly in the following assay for activity.

[0220] X Demonstration of TRANAC Activity

[0221] TRANAC can be expressed by transforming a mammalian cell linesuch as COS7, HeLa or CHO with an eukaryotic expression vector encodingTRANAC. Eukaryotic expression vectors are commercially available, andthe techniques to introduce them into cells are well known to thoseskilled in the art. The cells are incubated for 48-72 hours aftertransformation under conditions appropriate for the cell line to allowexpression of TRANAC. Then, phase microscopy is used to compare themitotic index of transformed versus control cells. A decrease in themitotic index indicates TRANAC activity.

[0222] XI Production of TRANAC Specific Antibodies

[0223] TRANAC that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Selection of appropriateepitopes, such as those near the C-terminus or in hydrophilic regions,is described by Ausubel et al. (supra), and others.

[0224] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems Model 431A peptide synthesizer(Perkin Elmer) using fmoc-chemistry, and coupled to keyhole limpethemocyanin (KLH, Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radio iodinated, goat anti-rabbit IgG.

[0225] XII Purification of Naturally Occurring TRANAC Using SpecificAntibodies

[0226] Naturally occurring or recombinant TRANAC is substantiallypurified by immunoaffinity chromatography using antibodies specific forTRANAC. An immunoaffinity column is constructed by covalently couplingTRANAC antibody to an activated chromatographic resin, such asCnBr-activated Sepharose (Pharmacia & Upjohn). After the coupling, theresin is blocked and washed according to the manufacturer'sinstructions.

[0227] Media containing TRANAC is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of TRANAC (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/TRANAC binding (eg, a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and TRANAC iscollected.

[0228] XIII Identification of Molecules Which Interact with TRANAC

[0229] TRANAC or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled TRANAC, washed and any wells withlabeled TRANAC complex are assayed. Data obtained using differentconcentrations of TRANAC are used to calculate values for the number,affinity, and association of TRANAC with the candidate molecules.

[0230] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 4 100 amino acids amino acid single linear BSTMNOT01 805296 1 Met SerThr Ser Thr Ser Cys Pro Ile Pro Gly Gly Arg Asp Gln Leu 1 5 10 15 ProAsp Cys Tyr Ser Thr Thr Pro Gly Gly Thr Leu Tyr Gly Thr Thr 20 25 30 ProGly Gly Thr Arg Ile Ile Tyr Asp Arg Lys Phe Leu Leu Glu Cys 35 40 45 LysAsn Ser Pro Ile Ala Arg Thr Thr Pro Cys Cys Leu Pro Gln Ile 50 55 60 ProGly Val Thr Thr Pro Pro Thr Ala Pro Leu Ser Lys Leu Glu Glu 65 70 75 80Leu Lys Glu Gln Glu Thr Glu Glu Glu Ile Pro Asp Asp Ala Gln Phe 85 90 95Glu Met Asp Ile 100 432 base pairs nucleic acid single linear BSTMNOT01805296 2 CTCCTCGACC TCAACGCCAG GCGGTTACTT TGCTGCTCCT NCCGCTCGCTATGTCAACGT 60 CCACTAGCTG CCCGATTCCC GGGGGCCGGG ACCAGCTGCC CGACTGCTACAGCACCACGC 120 CGGGGGGCAC GCTATACGGC ACTACCCCCG GAGGCACCAG GATCATCTACGACCGAAAGT 180 TCCTGCTGGA GTGCAAGAAC TCACCCATTG CCCGGACAAC CCCCTGCTGCCTCCCTCAGA 240 TTCCCGGGGT CACAACTCCT CCAACAGCCC CTCTYTCCAA GCTGGAGGAGCTGAAGGAGC 300 AGGAGACAGA GGAAGAGATA CCCGATGACG CACAATTTGA AATGGACATCTAATCCAGTG 360 CAGATGACCT GGCATGTGGA GTTACAGAGG GATCCCTCAT GCCACTGCTGCCACCACCTC 420 TTCCTGGGGC AT 432 120 amino acids amino acid singlelinear GenBank 561632 3 Met Ser Ser Ser Ala Gly Ser Gly His Gln Pro SerGln Ser Arg Ala 1 5 10 15 Ile Pro Thr Arg Thr Val Ala Ile Ser Asp AlaAla Gln Leu Pro His 20 25 30 Asp Tyr Cys Thr Thr Pro Gly Gly Thr Leu PheSer Thr Thr Pro Gly 35 40 45 Gly Thr Arg Ile Ile Tyr Asp Arg Lys Phe LeuLeu Asp Arg Arg Asn 50 55 60 Ser Pro Met Ala Gln Thr Pro Pro Cys His LeuPro Asn Ile Pro Gly 65 70 75 80 Val Thr Ser Pro Gly Thr Leu Ile Glu AspSer Lys Val Glu Val Asn 85 90 95 Asn Leu Asn Asn Leu Asn Asn His Asp ArgLys His Ala Val Gly Asp 100 105 110 Asp Ala Gln Phe Glu Met Asp Ile 115120 120 amino acids amino acid single linear GenBank 1658516 4 Met SerAla Ser Ala Gly Gly Ser His Gln Pro Ser Gln Ser Arg Ala 1 5 10 15 IlePro Thr Arg Thr Val Ala Ile Ser Asp Ala Ala Gln Leu Pro Gln 20 25 30 AspTyr Cys Thr Thr Pro Gly Gly Thr Leu Phe Ser Thr Thr Pro Gly 35 40 45 GlyThr Arg Ile Ile Tyr Asp Arg Lys Phe Leu Leu Asp Arg Arg Asn 50 55 60 SerPro Met Ala Gln Thr Pro Pro Cys His Leu Pro Asn Ile Pro Gly 65 70 75 80Val Thr Ser Pro Gly Ala Leu Ile Glu Asp Ser Lys Val Glu Val Asn 85 90 95Asn Leu Asn Asn Leu Asn Asn His Asp Arg Lys His Ala Val Gly Asp 100 105110 Glu Ala Gln Phe Glu Met Asp Ile 115 120

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:1, c) a biologically active fragment of a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1.
 2. Anisolated polypeptide of claim 1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1.
 3. An isolatedpolynucleotide encoding a polypeptide of claim
 1. 4. An isolatedpolynucleotide encoding a polypeptide of claim
 2. 5. An isolatedpolynucleotide of claim 4 comprising a polynucleotide sequence selectedfrom the group consisting of SEQ ID NO:2.
 6. A recombinantpolynucleotide comprising a promoter sequence operably linked to apolynucleotide of claim
 3. 7. A cell transformed with a recombinantpolynucleotide of claim
 6. 8. A transgenic organism comprising arecombinant polynucleotide of claim
 6. 9. A method of producing apolypeptide of claim 1, the method comprising: a) culturing a cell underconditions suitable for expression of the polypeptide, wherein said cellis transformed with a recombinant polynucleotide, and said recombinantpolynucleotide comprises a promoter sequence operably linked to apolynucleotide encoding the polypeptide of claim 1, and b) recoveringthe polypeptide so expressed.
 10. A method of claim 9, wherein thepolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:1.
 11. An isolated antibody which specificallybinds to a polypeptide of claim
 1. 12. An isolated polynucleotideselected from the group consisting of: a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:2, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical to a polynucleotidesequence selected from the group consisting of SEQ ID NO:2, c) apolynucleotide complementary to a polynucleotide of a), d) apolynucleotide complementary to a polynucleotide of b), and e) an RNAequivalent of a)-d).
 13. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A methodof detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 12, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 15. A method of claim 14, wherein the probe comprises atleast 60 contiguous nucleotides.
 16. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO:1.
 19. A methodfor treating a disease or condition associated with decreased expressionof functional TRANAC, comprising administering to a patient in need ofsuch treatment the composition of claim
 17. 20. A method of screening acompound for effectiveness as an agonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting agonist activity in the sample.21. A composition comprising an agonist compound identified by a methodof claim 20 and a pharmaceutically acceptable excipient.
 22. A methodfor treating a disease or condition associated with decreased expressionof functional TRANAC, comprising administering to a patient in need ofsuch treatment a composition of claim
 21. 23. A method of screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional TRANAC, comprising administering to apatient in need of such treatment a composition of claim
 24. 26. Amethod of screening for a compound that specifically binds to thepolypeptide of claim 1, the method comprising: a) combining thepolypeptide of claim 1 with at least one test compound under suitableconditions, and b) detecting binding of the polypeptide of claim 1 tothe test compound, thereby identifying a compound that specificallybinds to the polypeptide of claim
 1. 27. A method of screening for acompound that modulates the activity of the polypeptide of claim 1, themethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a sequence of claim 5, the method comprising:a) exposing a sample comprising the target polynucleotide to a compound,under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 29. A method of assessing toxicity of atest compound, the method comprising: a) treating a biological samplecontaining nucleic acids with the test compound, b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 12 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of TRANAC in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of TRANAC in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofTRANAC in a subject, comprising administering to said subject aneffective amount of the composition of claim
 34. 36. A method ofpreparing a polyclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, or an immunogenic fragment thereof, underconditions to elicit an antibody response, b) isolating antibodies fromsaid animal, and c) screening the isolated antibodies with thepolypeptide, thereby identifying a polyclonal antibody whichspecifically binds to a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1.
 37. A polyclonalantibody produced by a method of claim
 36. 38. A composition comprisingthe polyclonal antibody of claim 37 and a suitable carrier.
 39. A methodof making a monoclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, or an immunogenic fragment thereof, underconditions to elicit an antibody response, b) isolating antibodyproducing cells from the animal, c) fusing the antibody producing cellswith immortalized cells to form monoclonal antibody-producing hybridomacells, d) culturing the hybridoma cells, and e) isolating from theculture monoclonal antibody which specifically binds to a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1.
 40. A monoclonal antibody produced by a method of claim 39.41. A composition comprising the monoclonal antibody of claim 40 and asuitable carrier.
 42. The antibody of claim 11, wherein the antibody isproduced by screening a Fab expression library.
 43. The antibody ofclaim 11, wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 44. A method of detecting a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1 in a sample, the method comprising: a) incubating theantibody of claim 11 with a sample under conditions to allow specificbinding of the antibody and the polypeptide, and b) detecting specificbinding, wherein specific binding indicates the presence of apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1 in the sample.
 45. A method of purifying apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1 from a sample, the method comprising: a)incubating the antibody of claim 11 with a sample under conditions toallow specific binding of the antibody and the polypeptide, and b)separating the antibody from the sample and obtaining the purifiedpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1.
 46. A microarray wherein at least one elementof the microarray is a polynucleotide of claim
 13. 47. A method ofgenerating an expression profile of a sample which containspolynucleotides, the method comprising: a) labeling the polynucleotidesof the sample, b) contacting the elements of the microarray of claim 46with the labeled polynucleotides of the sample under conditions suitablefor the formation of a hybridization complex, and c) quantifying theexpression of the polynucleotides in the sample.
 48. An array comprisingdifferent nucleotide molecules affixed in distinct physical locations ona solid substrate, wherein at least one of said nucleotide moleculescomprises a first oligonucleotide or polynucleotide sequencespecifically hybridizable with at least 30 contiguous nucleotides of atarget polynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 57. A polynucleotide of claim 12, comprisingthe polynucleotide sequence of SEQ ID NO:2.